We propose to continue to molecularly clarify specific roles of PARP in radiation (IR)-induced apoptosis. It is based on the overall hypothesis that the covalent p(ADP-ribose)n of proteins by long, negatively charged chains of polymer renders target DNA-binding proteins "DNA phobic" and prevents their binding to DNA. Specific Aim 1 focuses on the regulation of human Ca2+/Mg2+ DNASIL3 during apoptosis, which causes late nucleosomal degradation of genomic DNA. DNASIL3 is inhibited and hence regulated in vitro when in the p(ADP-ribose)n state. Having successfully cloned the human DNASIL3, we wish to clarify its role in vivo via PARP regulation in IR-induced cell death. Cells transfected with a caspase-resistant PARP will be used to assess the consequences during IR-induced apoptosis of a now stable, uncleaved polymer, covalently bound to DNASIL3. Approaches in Specific Aim 2 will establish cell lines, stably transfected with inducible antisense and sense cDNA for PARP glycohydrolase (PARG). This enzyme specifically cleaves PAR from proteins such as DNAS1L3 and p53. Accordingly, PARG will be either eliminated or over expressed in cells. In the initial case we will test how the lack of ability to cleave PAR from DNAS1L3 and p53 will influence the regulation imposed by p(ADP-R)n on nuclear proteins. Complimentary PARG overexpression experiments will be performed to test the effects of a stable p(ADP-R)n state of the DNASIL3 and p53. Specific Aim 3 will focus on DFF45, an inhibitor of the nuclease, DFF40. This nuclease plays a key role in chromatin restructuring by 50 Kb DNA cleavage early in apoptosis. Cells derived from DFF45-/- and +/+ mice will be used to establish the mechanism underlying the protection of DFF45-/- cells against strand breaks-induced cell death. This involves key apoptotic events and a PARP-1-associated amplification loop. We will test whether IR causes a biphasic activation of PARP-1 during radiation-induced apoptosis immediately after direct DNA damage and secondly after DFF activation and the generation of 50 kb DNA breaks.